Insertion sequence element derived from Ralstonia solanacearum

ABSTRACT

The present invention provides three insertion elements and transposases encoded by the insertion elements that are derived from the genome of Ralstonia solanacearum, which has been isolated with a transposon trap vector.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.09/790,045, filed Feb. 21, 2001 U.S. Pat. No. 6,492,510.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a transposable element isolated fromthe genome of Ralstonia solanacearum and a transposase encoded by thetransposable element.

2. Description of the Related Art

It is known that transposable elements not only inactivate or activategenes before and after the transposition site by moving on the genome,but also cause various genome rearrangements such as deletion, inversionand duplication. It is also known that the transposable elementscontribute to genome plasticity that is important for the evolution andthe environmental adaptation of microorganisms (Arber et al., FEMSMicrob. Ecol., 15: 5-14 (1994)).

Transposable elements can be classified into transposons and insertionsequence elements. Transposons are defined as genes involved intransposition and having phenotypic genes such as antibioticresistances. On the other hand, insertion sequence elements(hereinafter, referred to as IS elements) are defined as genes thatgenerally have a size of 2 kb or less and have no phenotypic genes otherthan the genes involved in gene transposition (Gay et al., J.Bacteriol., 164: 918-921 (1985)).

The IS element was first discovered in the late 1960's as an elementthat causes mutation of E. coli. Thereafter, the IS elements of E. coliin particular were isolated and characterized. In the late 1980's, therewere reports of isolation of the IS elements from bacteria other thanthe E. coli group. At present, about 500 IS elements are reported tohave been isolated from medical bacteria, environmental bacteria andothers (Mahillon et al., Microbiol. Mol. Bio. Rev., 62:725-774(1998)).

The isolated IS elements are compared with each other based on the basesequence and the deduced amino acid sequence. The IS elements that havebeen found so far are classified into 17 families. Among these families,the IS3 family and the IS5 family are large families, and IS elementsbelonging to these families are isolated from bacteria ranging widelyfrom Gram-negative bacteria to Gram-positive bacteria (Mahillon et al.,ibid.).

The structure of the IS elements in the IS3 and the IS5 families ischaracterized by the presence of inverted repeat sequences at theirterminals and the inclusion of two open reading frames encoding atransposase. It seems that the terminal inverted repeat sequences playan important role in transposition, because recombination occurs inthese portions. For example, in insertion, 2 to 13 base pairs in thesite where insertion occurs are overlapped as same direct repeatsequences on both sides of the IS element (Galas and Chandler, MobileDNA, 109-162, ASM Press (Washington, D.C.) 1989).

Transposase is an enzyme that catalyzes an insertion reaction of a gene.In the IS elements of the IS3 family and the IS5 family, the transposaseis encoded by two open reading frames and is expressed as one protein intranslation. For example, in the case of the IS3 family, two openreading frames overlap, and a frame shift occurs in the overlappingportion. Then, the two open reading frames are translated successivelyso that the transposase is expressed (Ohtsubo and Sekine, Current topicsin Microbiology and Immunology, 204:1-26 (1996)).

Each of the IS elements obtained from various microorganisms has asequence that is unique to the particular microorganism, although theyare homologous. Utilizing the unique sequence, in the industrial fieldsof agriculture, foods, pharmacy and the like in which microorganisms areinvolved, the IS elements are used, for example, for identification ofplant pathogenic bacteria, diagnosis of infectious diseases,determination of infection routes and isolation of useful genes. The ISelement that is most common in practical use is IS6110, which isobtained from Mycobacterium tuberculosis and contributes toepidemiological surveys such as diagnosis of tuberculosis anddetermination of the infection route (Otal et al., J. Clin. Microbiol.,29:1252-1254 (1991)). Similar attempts have been conducted with variousbacteria.

IS elements are characterized by being able to activate or inactivategenes before and after the inserted site by transposition. Utilizingthis function positively, it is attempted to use the IS elements as atool for isolating useful genes (Haas et al., Molecular Biology ofPseudomonas, 238-249, ASM Press (Washington D.C.) (1996)).

The isolation of the IS elements was not conducted strategically, butmostly was rather accidentally achieved by analysis of mutants. This isbecause, as seen from the definition, IS elements have no phenotypeother than involvement in gene transposition, so that positive isolationwas difficult. Various transposon trap vectors have been developed sincepositive selection of the transposable element (a method of utilizingactivation of a detectable marker by transposition of the transposableelement to a part of a structural gene) was successfully reported in1985 (Gay et al., ibid.), and the IS elements have been isolated frombacteria such as Agrobacterium tumefaciens, Pseudomonas cepacia,Rhizobium meliloti, Rhizobium legumino sarum or the like by this method.

However, only 20 IS elements were isolated by this method, and this is asmall number relative to the total number of the isolated IS elements.The isolation of new IS elements from various bacteria is expected to beaccelerated as the positive selection is developed.

In regard to agriculturally important plant-related bacteria, Hasebe etal. isolated three kinds of IS elements from Pseudomonas glumae, whichis a common pathogenic bacterium in rice plants in Japan, by utilizing atransposon trap vector pSHI1063 (Hasebe et al., Plasmid, 39:196-204(1998)) (see Japanese Laid-Open Publication No. 10-248573). However, inreality, isolation of other IS elements has not been substantiallyresearched. Isolation of IS elements of various plant pathogenicbacteria (microorganisms) makes it possible to counter the pathogenicbacteria, so that there is a demand for isolation and utilization of theIS elements in the field of agriculture.

SUMMARY OF THE INVENTION

Therefore, with the foregoing in mind, it is an object of the presentinvention to provide new IS elements derived from Ralstoniasolanacearum, which is one of the common pathogenic bacteria invegetables. The Ralstonia solanacearum is a worldwide common soilinfectious plant pathogenic bacterium that causes wilt vessel disease tomore than 100 species of more than 30 families including Solanaceaeplants such as tomatoes, tobacco and potatoes. It is another object ofthe present invention to provide transposases that are encoded by theseIS elements. These IS elements and the transposases make it possible toprevent infection to Ralstonia solanacearum, for example, by effectingrecombination so that the transposases are inactivated, which is veryuseful in the field of agriculture.

In order to achieve the above objects, the inventors of the presentinvention have succeeded in isolating new IS elements from Ralstoniasolanacearum and realized the present invention.

The present invention provides an insertion sequence element or afunctional equivalent thereof comprising: a base sequence of SequenceI.D. No.2 at the 5′ terminal and a base sequence of Sequence I.D. No.3at the 3′.terminal as terminal inverted repeat sequences; and a basesequence encoding amino acid sequences of Sequence I.D. Nos.4 and 5 asopen reading frames between the terminal inverted repeat sequences.Herein, the open reading frame can be present overlapped orindependently.

The present invention further provides an insertion sequence elementconsisting of the base sequence of Sequence I.D. No.1.

Furthermore, the present invention provides an insertion sequenceelement or a functional equivalent thereof comprising: a base sequenceof Sequence I.D. No.7 at the 5′ terminal and a base sequence of SequenceI.D. No.8 at the 3′ terminal as terminal inverted repeat sequences; anda base sequence encoding amino acid sequences of Sequence I.D. Nos.9 and10 as open reading frames between the terminal inverted repeatsequences. Herein, the open reading frame can be present overlapped orindependently.

The present invention further provides an insertion sequence elementconsisting of a base sequence of Sequence I.D. No.6.

Furthermore, the present invention provides an insertion sequenceelement or a functional equivalent thereof comprising: a base sequenceof Sequence I.D. No.12 at the 5′ terminal and a base sequence ofSequence I.D. No.13 at the 3′ terminal as terminal inverted repeatsequences; and a base sequence encoding an amino acid sequence ofSequence I.D. No.14 as an open reading frame between the terminalinverted repeat sequences.

The present invention further provides an insertion sequence elementconsisting of a base sequence of Sequence I.D. No.11.

The present invention further provides a transposase or a functionalequivalent thereof expressed from a base sequence of positions 56 to 855of Sequence I.D. No. 1.

The present invention further provides a transposase or a functionalequivalent thereof expressed from a base sequence of positions 65 to 822of Sequence I.D. No.6.

The present invention further provides a transposase or a functionalequivalent thereof expressed from a base sequence of positions 44 to 865of Sequence I.D. No.11.

It should be noted that “movable genetic elements” (transposableelements) such as transposons may be significantly involved in theevolution and the environmental adaptation of organisms asself-mechanism of self-alternation of the organism's genome. The gene ofthe present invention moves on the genome of microorganisms and has thenature of activating or inactivating the gene that is positioneddownstream of the genome into which the gene of the present inventionjumps. Utilizing this property, it is possible to isolate industriallyuseful genes efficiently. Furthermore, utilizing these genes, it ispossible to determine the infection route and identify the bacteria inregard to Ralstonia solanacearum. Furthermore, the transpositionfunction of the transposable element is promoted by utilizing thetransposases of the present invention, so that it is possible to isolateindustrially useful genes efficiently. In addition, it is expected thatprotectant effects such as promotion of a reduction in the pathogenicitycaused by the mutation induction of Ralstonia solanacearum can beprovided.

These and other advantages of the present invention will become apparentto those skilled in the art upon reading and understanding the followingdetailed description with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing insertion positions of insertion sequencesof 26 clones.

FIG. 2 is a diagram showing the positional relationship betweenISJsp104.2 and an incomplete inverted sequence, a targeted overlappingsequence, ORFA and ORFB (SEQ ID NO: 1, with an additional TTA at eachend, and SEQ ID NOS: 4-5).

FIG. 3 is a diagram showing the positional relationship between ISmsp4.2and an incomplete inverted sequence, a targeted overlapping sequence,ORFA and ORFB (SEQ ID NO: 6, with an additional TAA at each end, and SEQID NOS: 9-10).

FIG. 4 is a diagram showing the positional relationship betweenISmsp101.3 and an incomplete inverted sequence, a targeted overlappingsequence and ORFA (SEQ ID NO: 11, with an additional TAA at each end,and SEQ ID NO:14).

DESCRIPTION OF THE PREFERRED EMBODIMENT (1) Definitions

In the present invention, “IS element” refers to a gene unit that has asize of 2 kb or less, has no phenotypic gene other than genes involvedin gene transposition, and includes one or two open reading framesencoding transposase and terminal inverted repeat sequences.

ISJsp104.2 is an IS element consisting of the base sequence of SequenceI.D. No.1.

ISmsp4.2 is an IS element consisting of the base sequence of SequenceI.D. No.6.

ISmsp101.3 is an IS element consisting of the base sequence of SequenceI.D. No.11.

In the present invention, “transposase” is an enzyme that catalyzes aninsertion reaction of a gene.

A “functional equivalent” used in the present invention refers to an ISelement or a transposase that substantially has the function or theactivity of the original IS element or transposase, and has at least90%, preferably at least 95% of homology in the base sequence or theamino sequence, respectively, when optimally aligned with the originalIS element or transposase.

Such a functional equivalent of the IS element includes substitution,addition, deletion or insertion of at least one nucleotide, in additionto the original sequence in the terminal inverted repeat sequence or theopen reading frame that is a functional site, and has at least functionsor activities substantially equivalent to those of the original ISelement or transposase. Examples of such a functional equivalent includeIS elements having a nucleotide substitution that causes conservativesubstitution of the amino acid of the transposase to be encoded, and ISelements having an intervening nucleotide in the open reading frame.

Such a functional equivalent of transposase may include substitution ofat least one amino acid (preferably conservative substitution), oradditional amino acid (e.g., a reader sequence, a secretion sequence,and a sequence that would advantageously function in purification), inaddition to the original sequence. It is appreciated that production ofthese functional equivalents is within a scope of technical knowledgethat can be routinely obtained by those skilled in the art.

(2) Method for Searching a Transposable Element

In the search of a transposable element, molecular biological experimenttechniques (electrophoresis of DNA, collection of electrophoresed DNAfrom a gel, digestion of restriction enzyme, PCR, labeling of DNA,hybridization, base sequencing and the like) can be used. Examples ofthese techniques include the techniques described in Sambrook et al., ALaboratory Manual, the second edition, Cold Spring Harbor Press, ColdSpring Harbor, N.Y. (1989) and other methods routinely used by thoseskilled in the art.

(a) Bacteria to be Used

In the present invention, Ralstonia solanacearum is used. A preferablestrain is a Ralstonia solanacearum strain MAFF301556. This strain is aRalstonia solanacearum isolated from a potato in Nagasaki in 1983. Thisstrain is deposited as a distributable strain with the Gene Bank of theMinistry of Agriculture, Forestry and Fisheries (2-1-2, Kannondai,Tsukuba-shi, Ibaraki) and is available to anyone for test and research.

(b) Principle

A transposon trap vector is used as means for isolating a transposableelement. In the present invention, pSHI1063 (Iida et al., Abstract ofProceeding of The fourteenth Conference of Molecule Biology Society ofJapan, p.216 (1991)) is used as the transposon trap vector. The trapvector pSHI1063 has a full length of 11.5 kb, and is a fusion plasmid ofthe plasmid pVS1 with wide host range of Pseudomonas aeruginosa and theplasmid pBR322 of E. coli. The trap vector pSHI1063 can be grown in awide range of Gram-negative bacteria such as E. coli, bacteria of thePseudomonas genus, the Agrobacterium genus, the Rhizobium genus and thelike. The trap vector pSHI1063 has an ampicillin-resistant gene and aspectinomycin-resistant gene as selective marker genes. In addition, inorder to trap a transposable element, this trap vector has a cIrepressor gene of λ phage and a kanamycin-resistant gene (neo) connectedto a P_(R) promoter that is under control of the cI repressor (this iscalled a trap cassette gene). After introducing the trap vector pSHI1063to a bacterium, a transposable element such as an IS element of thebacterium transposes into the cI repressor gene in the trap vectorpSHI1063. Then, the cI repressor in the trap vector is inactivated andthe P_(R) promoter is activated, so that the kanamycin-resistant geneoperates. Therefore, when the transposable element is present in the cIrepressor gene of the trap vector pSHI1063, the bacterium is resistantto kanamycin, and therefore the transposable element can be selectedefficiently.

(c) Acquisition of the Transposable Element

Based on the above principle, the trap vector pSHI1063 is introduced toa Ralstonia solanacearum strain MAFF301556, and a selectedkanamycin-resistant strain is cultured, and boiled, for example, at 100°C. for 5 minutes so that the bacterial cells are lysed, and the DNA isextracted. Then, PCR is performed using the sequence of the trapcassette gene as the primer so as to amplify a DNA fragment having atransposable element (Sambrook et al., ibid.).

(d) Base Sequencing of the Transposable Element

Next, the base sequence of the amplified and collected insertionsequence is determined. First, an intended DNA fragment is prepared froma product of the PCR amplification including the transposable elementobtained in (c), and is subcloned into an appropriate vector, forexample, a pT7Blue vector. A plasmid DNA is prepared from the obtainedpositive clone, and is used as a template for base sequencing. Thus,this plasmid DNA is sequenced on both strands, for example, by primerwalking method. For sequencing, an autosequencer (model 1377manufactured by ABI Corp.) can be used for example.

(e) Homology Search of the Isolated Transposable Element

A database (DNA Information and Stock Center, URL=is searched for thebase sequence homology and the amino acid homology of the base sequencedtransposable element, and a novel gene is characterized. In this manner,the transposable element is selected.

EXAMPLES

Hereinafter, the present invention will be described by way of examples.

Example 1

Search for a transposable element of a Ralstonia solanacearum strainMAFF301556 (hereinafter, referred to simply as “strain MAFF301556”) wasperformed. To search the transposable element, a transposon trap vectoris required, and in the present invention, pSHI1063 was used.

First, the strain MAFF301556 was grown on PTYG agar medium (0.25 g ofbactopeptone, 0.25 g of bactotryptone, 0.5 g of bacto-yeast extract, 0.5g of glucose, 30 mM of MgSO_(4.)7H₂O, 3.5 mg of CaCl_(2.)2H₂O, and 15 gof agar powder in 1000 ml of distilled water) at 28° C. The grown colonywas inoculated into a 5 ml of a PTYG liquid medium and was cultured withshaking at 28° C. for 2 days.

Competent cells were made from this culture solution. Four ml of theculture solution were added to 200 ml of the PTYG medium, andcultivation was performed with shaking at 28° C. until the OD₅₀₀ reachesabout 0.2. After cultivation, the culture solution was placed in ice for20 minutes and then centrifuged (3000 rpm/10 min/4° C.) so as to collectbacterial pellets. The pellets were suspended in 10 ml of an ice-cold1mM HEPES.NaOH buffer (pH 7.0). Then, the suspension was centrifugedagain to collect bacterial pellets, and the pellets were suspended in 10ml of an ice-cold 1 mM HEPES.NaOH buffer (pH 7.0). Then, the bacterialpellets were collected by centrifugation (8000 rpm/10 min/4° C.), andwere suspended in 2 ml of an ice-cold 10% glycerol solution, so thatcompetent cells were prepared.

Then, 40 μl of the obtained competent cells were mixed with about 0.36μg of a transposon trap vector pSHI1063 (a liquid volume of 1 μl)dissolved in a TE buffer (10 mM Tris.HCl (pH8.0)−1 mM EDTA) and themixture was transferred to a cuvette that was previously cooled to 4° C.Electric pulses were applied to this cuvette with a high voltage pulsegenerator, “Gene Pulser” manufactured by Biorad Co. The parameters forthe electric pulse were an electrical resistance of 200Ω, an electricalcapacity of 25 μFD, and an electric field of 6.25 KV/cm.

After applying pulses, a cell/DNA mixture was immediately transferred to2 ml of the PTYG medium, and was cultured with shaking at 28° C. for onehour, followed by centrifugation (8000 rpm/10 min/4° C.). Then,bacterial pellets were suspended in 1 ml of sterilized distilled water.The suspension was serial diluted by a factor of 10 with sterilizeddistilled water sequentially, and diluted solutions with bacterial cellsof up to 10⁻⁴ were obtained. Then, 100 μl of the diluted solutions wereplated onto PIYG agar plates containing 100 μg/ml of spectinomycin.Further, the same number of bacterial cells was plated onto a PTYG agarplate that did not contain any antibodies as the control to check thetransformation frequency, followed by culturing at 28° C. for 2 days.The transformed colonies resistant to spectinomycin that appeared wereapplied to a PIYG plate containing spectinomycin again with a platinumloop, and were cultured at 28° C. for 2 days, so that a single colonywas formed. The transformation frequency of pSHI1063 to the Ralstoniasolanacearum strain MAFF 301556 was 5×10⁻⁴.

Next, the spectinomycin-resistant colonies that formed a single colonywere picked up one by one with a platinum loop and inoculated into 5 mlof a PTYG medium containing 100 μg/ml of spectinomycin, followed byculturing with shaking at 28° C. for 2 days. After cultivation, thesuspension was serially diluted by a factor of 10 with sterilizeddistilled water. Then, 100 μl of the diluted solutions with thebacterial cells ranging from an undiluted solution of the suspension toa 10⁻⁶ diluted solution were applied onto PTYG agar plates containing 50μg/ml of kanamycin and 100 μg/ml of spectinomycin. Further, the samenumber of bacterial cells was plated onto a PTYG agar plate containing100 μg/ml of spectinomycin as the control to check the appearancefrequency of mutants, followed by culturing at 28° C. for 2 days. Theobtained transformed colonies resistant to kanamycin were applied to aPTYG plate containing 50 μg/ml of kanamycin and 100 μg/ml ofspectinomycin again with a platinum loop, so that a single colony wasformed.

The appearance frequency of mutants from the obtained transformantsranged widely from about 100% to 6×10⁻⁷, and was significantly differentdepending on the transformed clone. Ninety-seven mutants obtained fromthese transformants were used for the following examinations.

With respect to the 97 mutants, the DNA fragments of the insertionsequence were amplified by a PCR technique using various primersprepared based on the trap cassette gene sequence of pSHI1063 by routineprocedures. For amplification, using a DNA amplifier (PC800 manufacturedby ASTEC Inc.), a cycle of DNA denaturation at 94° C. for 1 minute,annealing at 55° C. for 2 minutes, and DNA elongation at 72° C. for 3minute was repeated 25 times. After a PCR reaction, 1 μl of the reactionsolution was used for agarose electrophoresis (1.5% agarose) to confirmthe DNA amplification.

As a result, it was confirmed that DNA insertion was not observed in theplasmids of 71 kanamycin resistant mutants, whereas DNA fragments wereinserted in the trap vectors pSHI1063 in the plasmids obtained from 26mutants. The trap cassette gene of pSHI1063 was divided into 6 regionsfrom the upstream to the downstream (divided into I to VI regions fromthe uppermost stream region of the cI gene to the upstream of the neogene), and it was investigated into which region the DNA was inserted.The results were region I for 3 mutants, region II for 14 mutants,region III for 2 mutants, region IV for 6 mutants, region V for 1mutant, and region VI for none (FIG. 1).

Next, the base sequences of the plasmids obtained from the 26 mutantswere determined. The PCR reaction products were subcloned into pT7Bluevectors. Plasmid DNAs were prepared from the obtained positive clonesand used as templates for base sequencing. These plasmid DNAs weresequenced on both strands, for example by primer walking with anautosequencer (model 1377 manufactured by ABI Corp.) (Sambrook et al.,ibid).

With respect to the sequenced DNA fragments, a public database (DNAInformation and Stock Center, was searched for base sequence homologyand amino acid sequence homology, and the genes were characterized.Table 1 shows the results.

TABLE 1 The characteristics of DNA inserted in the pSHI1063 trapcassette Insertion No. Clone Deduced size region IS element  1 Jsp104.2864 bp I ISJsp104.2  2 Jsp105.1 1.2 kb I iso-IS1420  3 Jsp101.5 1.2 kb Iiso-IS1420  4 msp101.3 884 bp II ISmsp101.3  5 Jsp104.1 1.3 kb II  6Jsp107.3 1.2 kb II iso-IS1420  7 Jsp107.4 1.2 kb II iso-IS1420  8Jsp105.5 1.2 kb II iso-IS1420  9 Jsp120.5 1.2 kb II iso-IS1420 10Jsp120.1 1.2 kb II iso-IS1420 11 Jsp101.2 1.2 kb II iso-IS1420 12Jsp102.3 1.0 kb II 13 Jsp101.4 1.2 kb II iso-IS1420 14 Jsp105.3 1.2 kbII iso-IS1420 15 Jsp120.2 1.2 kb II iso-IS1420 16 Jsp120.3 1.2 kb IIiso-IS1420 17 Jsp120.4 1.2 kb II iso-IS1420 18 msp4.4 0.8 kb III 19Jsp105.2 0.8 kb III 20 msp101.5 1131 bp IV IS 1420 21 msp 4.2 842 bp IVISmsp 4.2 22 Jsp102.1 1.2 kb IV iso-IS1420 23 Jsp107.1 1.2 kb IViso-IS1420 24 Jsp104.3 0.2 kb IV 25 Jsp105.4 1.2 kb IV iso-IS1420 26Jsp107.2 1.2 kb V iso-IS1420

As a result, three novel IS elements (ISJsp104.2, ISmsp4.2 andISmsp101.3) were isolated as the transposable elements. Their propertiesare as follows.

(a) ISJsp104.2

ISJsp104.2 is a base sequence with a full length of 864 bp composed ofthe base sequence of Sequence I.D. No.1, and has incomplete invertedrepeat sequences (19 bp) at its terminals (the underlined arrow portionof FIG. 2, Sequence I.D. Nos. 2 and 3). Targeted overlapping sequencesof 3 bp are coupled to both terminals of ISJsp104.2, and the sequencewas TTA (the squared portions in FIG. 2). In comparison with thehomology of the base sequence, ISJsp104.2 has a high homology of 74.8%with IS1418 (Burkholderia glumae), 70.9% with ISB111 (Ralstoniasolanacearum) and 59.5% with IS402 (Burkholderia cepacia), which are ISelements belonging to the IS427 subgroup of the IS5 family (Mahillon etal., ibid.). Therefore, it seems that ISJsp104.2 is a novel IS elementobtained from Ralstonia solanacearum that belongs to the IS427 subgroupof the IS5 family.

(Transposase Encoded by ISJsp104.2)

The base sequence analysis and the amino acid sequence homology analysisof ISJsp104.2 were performed and it was confirmed that this IS elementencoded transposase, as other IS elements. ISJsp104.2 has two openreading frames (ORFA, ORFB) that are believed to encode a transposase(transposable enzyme), as generally seen in the IS3 and the IS5 families(Mahillon et al., ibid.). These open reading frames partly overlap, andare frame shifted (FIG. 2). Furthermore, there is a characteristic motif(C₅G₆) that is estimated to be involved in the frame shift in the basesequence in the overlapped portion (the underlined portion in FIG. 2)(Iversen et al., Plasmid 32:46-54 (1994)). ORFA is composed of 134 aminoacids (Sequence I.D. No. 4) and ORFB is composed of 211 amino acids(Sequence I.D. No. 5). The ORFA and the ORFB of ISJsp104.2 havehomologies of at least 70% in the amino acid sequences with the ORFA andthe ORFB of IS 1418, respectively.

(b) ISmsp4.2

ISmsp4.2 is a base sequence with a full length of 842 bp composed ofSequence I.D. No.6, and has incomplete inverted repeat sequences (18 bp)at its terminals (the underlined arrow portion of FIG. 3, Sequence I.D.Nos. 7 and 8). Targeted overlapping sequences of 3 bp are coupled toboth terminals of ISmsp4.2, and the sequence is TAA (the squaredportions in FIG. 3). In comparison with the homology in the basesequence, ISmsp4.2 has a high homology of 56.7% with IS427(Agrobacterium tumefaciens) and 54.9% with IS298 (Caulobactercrescentus), which are IS elements belonging to the IS427 subgroup ofthe IS5 family (Mahillon et al., ibid.). Therefore, it seems thatISmsp4.2 is a novel IS element obtained from Ralstonia solanacearum thatbelongs to the IS427 subgroup of the IS5 family.

Both the ISmsp4.2 and the ISmsp104.2 belong to the IS427 subgroup of theIS5 family, but have a homology as low as 50% or less to each other.

(Transposase Encoded by ISmsp4.2)

Also ISmsp4.2 has two open reading frames, ORFA (116 amino acids)(Sequence I.D. No.9) and ORFB (159 amino acids) (Sequence I.D. No.10)that are believed to encode a transposase. As other IS elements thatbelong to the IS427 subgroup of the IS5 family, the ORFA and the ORFBpartly overlap, and are frame shifted (FIG. 3). Furthermore, there is acharacteristic motif (A₆G) that is estimated to be involved in the frameshift in the base sequence in the overlapped portion (the underlinedportion in FIG. 3) (Ohtsubo and Sekine, ibid.). The ORFA and the ORFB donot have a high homology in the amino acid sequence with other ISelements, and the homologies are 40% or less in any cases.

(c) ISmsp101.3

ISmsp101.3 is a base sequence with a full length of 884 bp composed ofSequence I.D. No.11, and has incomplete inverted repeat sequences (18bp) at its terminals (the underlined arrow portion of FIG. 4, SequenceI.D. Nos.12 and 13). Targeted overlapping sequences of 3 bp are coupledto both terminals of ISmsp101.3, and the sequence is TAA (the squaredportions in FIG. 4). In comparison with the homology in the basesequence, ISmsp101.3 has homologies of 67.6% with IS12528 (Gluconobactersuboxydans), 56.6% with ISR1F7-2 (Rhizobium leguminosarum), 56.5% withISRm220-12-1 (Sinorhizobium meliloti) and 54.6% with IS1031 (Acetobacterxylinum), which are IS elements belonging to the IS1031 subgroup of theIS5 family (Mahillon et al., ibid.). Therefore, it is believed thatISmsp101.3 is a novel IS element obtained from Ralstonia solanacearumthat belongs to the IS1031 subgroup of the IS5 family.

(Transposase encoded by ISmsp101.3)

Also ISmsp101.3 has an open reading frame, ORFA (274 amino acids)(Sequence I.D. No.14) that is believed to encode a transposase, and hasa high homology in the amino acid sequence of 71.1% with the ORFA274 ofIS12528, which belongs to the IS1031 subgroup of the IS5 family.

14 1 864 DNA Ralstonia solanacearum 1 gggccgctaa caaaaccaag tcatcgaacgcaggtggttg agcgttgttg ttggcatggc 60 acgaaagaag atcagcaatg aactgtggaaggcgttgcaa ccgctgctgc cggttgtgga 120 gccttcgacc aaaggcggtc gtccgcgcgtggatgatcgg gcggcgctga acggcatcct 180 gtttgttctg cataccggta tcccgtgggaagacctgcct aaagaactgg gctttggcag 240 cggcatgacg tgctggcgtc gcctgcgggagtggcaggcc aacggcgttt gggagcggct 300 gcatttggct ctgctcaagc gcctgcgcgaacacgaccag atcgactgga gccgagccag 360 tgtcgacggt gcaacggtgg ccagcccccggggggcgagc agacggggcc gaatccaacg 420 gatcgtggca agctcggtag caagcgccatctcgtcgtag atcggcgcgg cgtgccgttg 480 gcgctgatgg tcaccggtgc caatcgtcacgactcggtgg tgttcgaggt gctcgttgac 540 gccatcccga gcgtgcccgg actcgatggccgcccgcgat gccgccccga caagcttcac 600 gcggataagg gatacgactt cgcgcgatgccgccggcatc tgcgcaagcg gggcatgact 660 ccccggatcg ctcgccgtgg catcgagaagaacgaccggc tcggcaagca tcgctgggtt 720 gtcgagcgca cccatgcctg gcttgctggcttcggcaagt tgcgcattcg tttcgagcgt 780 tctcttcaga ctcatctcgc tttgctcaccctggcttgcg ccgtcatctg cgggcgattt 840 gttgatcggt tttgttagcg actc 864 2 19DNA Ralstonia solanacearum 2 gggccgctaa caaaaccaa 19 3 19 DNA Ralstoniasolanacearum 3 tcggttttgt tagcgactc 19 4 134 PRT Ralstonia solanacearum4 Met Ala Arg Lys Lys Ile Ser Asn Glu Leu Trp Lys Ala Leu Gln Pro 1 5 1015 Leu Leu Pro Val Val Glu Pro Ser Thr Lys Gly Gly Arg Pro Arg Val 20 2530 Asp Asp Arg Ala Ala Leu Asn Gly Ile Leu Phe Val Leu His Thr Gly 35 4045 Ile Pro Trp Glu Asp Leu Pro Lys Glu Leu Gly Phe Gly Ser Gly Met 50 5560 Thr Cys Trp Arg Arg Leu Arg Glu Trp Gln Ala Asn Gly Val Trp Glu 65 7075 80 Arg Leu His Leu Ala Leu Leu Lys Arg Leu Arg Glu His Asp Gln Ile 8590 95 Asp Trp Ser Arg Ala Ser Val Asp Gly Ala Thr Val Ala Ser Pro Arg100 105 110 Gly Ala Ser Arg Arg Gly Arg Ile Gln Arg Ile Val Ala Ser SerVal 115 120 125 Ala Ser Ala Ile Ser Ser 130 5 211 PRT Ralstoniasolanacearum 5 Arg Thr Gly Leu Trp Gln Arg His Asp Val Leu Ala Ser ProAla Gly 1 5 10 15 Val Ala Gly Gln Arg Arg Leu Gly Ala Ala Ala Phe GlySer Ala Gln 20 25 30 Ala Pro Ala Arg Thr Arg Pro Asp Arg Leu Glu Pro SerGln Cys Arg 35 40 45 Arg Cys Asn Gly Gly Gln Pro Pro Gly Gly Glu Gln ThrGly Pro Asn 50 55 60 Pro Thr Asp Arg Gly Lys Leu Gly Ser Lys Arg His LeuVal Val Asp 65 70 75 80 Arg Arg Gly Val Pro Leu Ala Leu Met Val Thr GlyAla Asn Arg His 85 90 95 Asp Ser Val Val Phe Glu Val Leu Val Asp Ala IlePro Ser Val Pro 100 105 110 Gly Leu Asp Gly Arg Pro Arg Cys Arg Pro AspLys Leu His Ala Asp 115 120 125 Lys Gly Tyr Asp Phe Ala Arg Cys Arg ArgHis Leu Arg Lys Arg Gly 130 135 140 Met Thr Pro Arg Ile Ala Arg Arg GlyIle Glu Lys Asn Asp Arg Leu 145 150 155 160 Gly Lys His Arg Trp Val ValGlu Arg Thr His Ala Trp Leu Ala Gly 165 170 175 Phe Gly Lys Leu Arg IleArg Phe Glu Arg Ser Leu Gln Thr His Leu 180 185 190 Ala Leu Leu Thr LeuAla Cys Ala Val Ile Cys Gly Arg Phe Val Asp 195 200 205 Arg Phe Cys 2106 842 DNA Ralstonia solanacearum CDS (65)...(822) 6 gggtcaggacccattgattt gaattgacgg ctatgattca gacgggcgga taggagcctg 60 actgatgagtaatttgttct ggctgactaa cgagcaaatg gctcgtcttc agccctattt 120 ccccaagagccatggccgcc agcgtgtcga tgatcggcgt gtgctgagcg gcatcatttt 180 cgtcaatcgcaacgggctcc ggtggtgcga tgcgccgaag gaatatggcc cggcgaaaac 240 gctgtataaccgctggaaac gctggagcga caagggcatc tttatccaga tgatggacgg 300 cctggctgtgcctgaagctg cagaacacca gaccgtcatg attgatgcaa cctatctcaa 360 ggcccaccgcacggcttcga gcctgcgggt aaaaaagggg gcgcgggtcg cctgattgga 420 cgcacgaaaggcgggatgaa caccaagctt catgccgtga cggatgcgag tggtcgcccg 480 atcagtttcttcataacggc cggtcaaatc agcgattaca ccggtgctgc cgccttgctt 540 gatgaacttcccaaggccaa atggctactg gccgaccgtg gctatgatgc cgactggtat 600 cgtgacgcgttacaggcgaa ggggatcact ccctgcattc ccggtcggaa atcccggacc 660 acgaccatcaaatacgacaa acgccgctat aaacggcgca accgaataga gatcatgttc 720 gggcgtctcaaggattggcg acgtgtcgct acgcgctatg acaggtgccc aatggctttt 780 ctttccgccatctctctcgc tgcaaccgtt atcttctggc tctgatcaac gagtcctgac 840 cc 842 7 17DNA Ralstonia solanacearum 7 gggtcaggac ccattga 17 8 17 DNA Ralstoniasolanacearum 8 tcaacgagtc ctgaccc 17 9 116 PRT Ralstonia solanacearum 9Met Ser Asn Leu Phe Trp Leu Thr Asn Glu Gln Met Ala Arg Leu Gln 1 5 1015 Pro Tyr Phe Pro Lys Ser His Gly Arg Gln Arg Val Asp Asp Arg Arg 20 2530 Val Leu Ser Gly Ile Ile Phe Val Asn Arg Asn Gly Leu Arg Trp Cys 35 4045 Asp Ala Pro Lys Glu Tyr Gly Pro Ala Lys Thr Leu Tyr Asn Arg Trp 50 5560 Lys Arg Trp Ser Asp Lys Gly Ile Phe Ile Gln Met Met Asp Gly Leu 65 7075 80 Ala Val Pro Glu Ala Ala Glu His Gln Thr Val Met Ile Asp Ala Thr 8590 95 Tyr Leu Lys Ala His Arg Thr Ala Ser Ser Leu Arg Val Lys Lys Gly100 105 110 Ala Arg Val Ala 115 10 159 PRT Ralstonia solanacearum 10 CysAsn Leu Ser Gln Gly Pro Pro His Gly Phe Glu Pro Ala Gly Lys 1 5 10 15Lys Gly Gly Ala Gly Arg Leu Ile Gly Arg Thr Lys Gly Gly Met Asn 20 25 30Thr Lys Leu His Ala Val Thr Asp Ala Ser Gly Arg Pro Ile Ser Phe 35 40 45Phe Ile Thr Ala Gly Gln Ile Ser Asp Tyr Thr Gly Ala Ala Ala Leu 50 55 60Leu Asp Glu Leu Pro Lys Ala Lys Trp Leu Leu Ala Asp Arg Gly Tyr 65 70 7580 Asp Ala Asp Trp Tyr Arg Asp Ala Leu Gln Ala Lys Gly Ile Thr Pro 85 9095 Cys Ile Pro Gly Arg Lys Ser Arg Thr Thr Thr Ile Lys Tyr Asp Lys 100105 110 Arg Arg Tyr Lys Arg Arg Asn Arg Ile Glu Ile Met Phe Gly Arg Leu115 120 125 Lys Asp Trp Arg Arg Val Ala Thr Arg Tyr Asp Arg Cys Pro MetAla 130 135 140 Phe Leu Ser Ala Ile Ser Leu Ala Ala Thr Val Ile Phe TrpLeu 145 150 155 11 884 DNA Ralstonia solanacearum CDS (44)...(865) 11gagcccgttt gaaaattccc cgccgttgtg gtgtaaaggc gggatgtgga aaaaagaaga 60tcgagagcgt gaggcgaagc tggctcggaa gaccaagcgt tacccgagcg acctgacgga 120tatcgaatgg gccgctgtgc agccgctgct gccacgcgcg gccgtgcgag gccggcgtcg 180ggagtgcgac ttgagggagg tggtcaacgc cttgcgctat ctggtgcgag cgggctgcgg 240ttggcgcatg ctgccgcacg acttcccgcc ttggcaaacc gtgtattggt ggtttcgtcg 300gctcatgcgt cgcttcctgt tccgcacgct gcacgacgtg gtgctgatgt tggaccggga 360gttggctggg cgccagccgt gcccgagtgc gggcgtcatc gacagccaga cagtcaaagc 420gccctcggcc gacaagcgtg gctacgacgc ggccaagaaa atcgtcgggc gcaagcggca 480tatcgcggtg gacacggatg gacggctgct gatggtgaac ctgacaccgg cggacattgc 540cgatagcacg ggtgcgctgg cggtgctgga ggcggtgaag aagcgctggc caggcataaa 600acacctgttc gctgacggtg cgtatgaccg cacaacgctg atggacaagg catcgaccct 660cgacttcgtg gttgaggtgg tgcgccggca cgagcagcaa acgggctttg ccgttctgcc 720gcgccgctgg gtggtcgagc gcacctttgg gtggatggtt cgctggcgcc gactcgtacg 780cgactacgag cagcgcgcgg acgtctcgga agccatgatt catatcgcga tgagcggctt 840gctactgcgc agaatcgctc atccttgaat ttccaaacgg gctc 884 12 18 DNA Ralstoniasolanacearum 12 gagcccgttt gaaaattc 18 13 18 DNA Ralstonia solanacearum13 gaatttccaa acgggctc 18 14 274 PRT Ralstonia solanacearum 14 Met TrpLys Lys Glu Asp Arg Glu Arg Glu Ala Lys Leu Ala Arg Lys 1 5 10 15 ThrLys Arg Tyr Pro Ser Asp Leu Thr Asp Ile Glu Trp Ala Ala Val 20 25 30 GlnPro Leu Leu Pro Arg Ala Ala Val Arg Gly Arg Arg Arg Glu Cys 35 40 45 AspLeu Arg Glu Val Val Asn Ala Leu Arg Tyr Leu Val Arg Ala Gly 50 55 60 CysGly Trp Arg Met Leu Pro His Asp Phe Pro Pro Trp Gln Thr Val 65 70 75 80Tyr Trp Trp Phe Arg Arg Leu Met Arg Arg Phe Leu Phe Arg Thr Leu 85 90 95His Asp Val Val Leu Met Leu Asp Arg Glu Leu Ala Gly Arg Gln Pro 100 105110 Cys Pro Ser Ala Gly Val Ile Asp Ser Gln Thr Val Lys Ala Pro Ser 115120 125 Ala Asp Lys Arg Gly Tyr Asp Ala Ala Lys Lys Ile Val Gly Arg Lys130 135 140 Arg His Ile Ala Val Asp Thr Asp Gly Arg Leu Leu Met Val AsnLeu 145 150 155 160 Thr Pro Ala Asp Ile Ala Asp Ser Thr Gly Ala Leu AlaVal Leu Glu 165 170 175 Ala Val Lys Lys Arg Trp Pro Gly Ile Lys His LeuPhe Ala Asp Gly 180 185 190 Ala Tyr Asp Arg Thr Thr Leu Met Asp Lys AlaSer Thr Leu Asp Phe 195 200 205 Val Val Glu Val Val Arg Arg His Glu GlnGln Thr Gly Phe Ala Val 210 215 220 Leu Pro Arg Arg Trp Val Val Glu ArgThr Phe Gly Trp Met Val Arg 225 230 235 240 Trp Arg Arg Leu Val Arg AspTyr Glu Gln Arg Ala Asp Val Ser Glu 245 250 255 Ala Met Ile His Ile AlaMet Ser Gly Leu Leu Leu Arg Arg Ile Ala 260 265 270 His Pro

What is claimed is:
 1. An isolated insertion sequence element or a functional equivalent thereof comprising: the base sequence of SEQ ID NO: 12 at a 5′ terminus and the base sequence of SEQ ID NO: 13 at a 3′ terminus as terminal inverted repeat sequences; and a base sequence encoding the amino acid sequence of SEQ ID NO: 14 as an open reading frame between the terminal inverted repeat sequences.
 2. An isolated insertion sequence element consisting of the base sequence of SEQ ID NO:
 11. 